The -KTS splice variant of WT1 is essential for ovarian determination in mice

Sex determination in mammals depends on the differentiation of the supporting lineage of the gonads into Sertoli or pre-granulosa cells that govern testis and ovary development, respectively. While the Y-linked testis-determining gene Sry has been identified, the ovarian-determining factor remains unknown. Here we identify -KTS, a major alternatively spliced isoform of the Wilms’ tumor suppressor WT1, as a key determinant of female sex determination. Loss of -KTS variants blocks gonadal differentiation in mice, whilst increased expression, as found in Frasier syndrome, induces precocious differentiation of ovaries independently of their genetic sex. In XY embryos, this antagonizes Sry expression, resulting in male-to-female sex reversal. Our results identify - KTS as an ovarian-determining factor and demonstrate that its time of activation is critical in gonadal sex differentiation.


Mouse strains and genotyping
All experiments described herein were conducted in compliance with the relevant institutional and French animal welfare laws and policies.Mouse lines were kept on a mixed background B6CBAF1/JRj.The Wt1 tm1Jae (referred to as D), KTS (-KTS -/-KTS -referred to as -KTS KO) and Frasier (+KTS -/+KTS -referred to as +KTS KO) lines have been described previously in (15,20).Heterozygous +KTS -/+ were mated to D/+ mice to obtain compound heterozygous referred to as +KTS KO/D embryos.The day of the presence of a vaginal plug was marked embryonic day 0.5 (E0.5).Embryos collected at E11.5-12.0 were staged by counting the number of tail somites (ts) with 18 ts corresponding to E11.5 (35).The day of delivery was defined as post-natal day 0 (P0).The genotyping was performed on lysates of tail tips or ear biopsies as described previously in (20,36).The primers are listed in Table S2.BAC Wt1-KTS transgenic line: BAC Wt1, RP24-67H19 (37) was obtained from the Children's Hospital Oakland Research Institute (CHORI, USA) and modified by recombineering techniques performed by Gen-H GmbH (Genetic Engineering Heidelberg) by insertion of the point mutation T->C in the second splice donor site at position +2 in intron 9 as described in (20).This promotes the expression of -KTS and prevents the synthesis of +KTS variants.The BAC was digested by NotI to obtain linear DNA and dialyzed against 10 mM Tris:HCl pH7.4,0.1 mM EDTA to a final concentration of 2 ng/µl (Fig. S9A).Microinjection was performed using fertilized oocytes as described in (37).The embryos were collected at E12.5 from the fosters, genotyped as described in (20), and gonads were processed for immunological analyses.Four XY transgenic embryos Tg(RP24-67H19; Wt1-KTS) were obtained with different degrees of mosaicism.The transgenic embryos denoted Tg(Wt1-KTS)14 and Tg(Wt1-KTS)41 are shown in Fig. 4A-B, Fig. S9B.

In situ hybridization
Tissues were fixed overnight at room temperature with 4% paraformaldehyde, processed for paraffin embedding, and sectioned at 5 µm thick.Rspo1 mRNA were detected using the RNAscope technology (479591 probe) and -KTS and +KTS variants using the Basescope Duplex Detection Kit (715291-C2 and 715281 probes, respectively).The protocol was performed according to Advanced Cell's instructions using the chromogenic Fast Red or Fast Green dye.Slides were counterstained with Hoechst, and images were obtained on microscopes (LSM780, AxioObservera and Axiovert 200M,Carl Zeiss).

Image analysis of Basescope in situ hybridization
The image acquisition was performed with a Axiovert 200M (Zeiss) with 10x/0.3objective equipped with an Andor Neo camera.From this setup, an analysis was carried out in 4 steps, called DICHisto.First, we developed in Metamorph (Molecular Devices, Sunnyvale, CA) a program to acquire sequentially, in DIC, the wavelength range 440/60, 525/50, and 605/70 corresponding to color camera RGB (but without Bayer demosaicing artefact).After an auto-calibration step (optimizing exposures for global white balance), the process could be inserted in multidimensional acquisition (Z, time, mosaic) and combined with fluorescence.Second, we developed in Matlab (The Mathworks, Natick US), a program to quantify the amount of Basescope staining of +KTS and -KTS staining from raw images.We corrected inhomogeneous illumination with a local white balance.After converting in HSL, we extracted "FHisto" signals corresponding to the saturation value filtered based on hue for +KTS and -KTS (and to a lesser extent the luminance).The luminance channel corresponds to the monochromatic DIC image.Then this generated a multi-channel stack including FHistos, DIC, and fluorescence channels (DAPI and others if any).Third, we developed a set of ImageJ macros that is designed for interactive whiteboard.We drew manually the nuclei of the gonad (a second category has been defined for indiscernible nuclei).Then the program quantified the statistics per image including the area of +KTS and -KTS per nucleus and in the cytoplasm.And fourth, we summarized all statistical parameters developing another Matlab program.All programs with test images are available (32).

Immunological analyses
Gonad samples were fixed, embedded, and sectioned as described for in situ hybridization.Sections were rehydrated, boiled in a pressure cooker for 2 min with Antigen Unmasking Solution (Vector laboratories), and blocked in PBS solution containing 10% normal donkey serum and 3% BSA.All antibodies were applied overnight at 4 °C at the concentrations listed in Table S3.Alexaconjugated secondary antibodies were diluted 1:200 and applied at room temperature for 1 h.Slides were counterstained with DAPI diluted in the mounting medium (Vectashield, Vector Laboratories).The images were generated with a motorized Axio Imager Z1 microscope (Zeiss) or with confocal laser scanning microscope (LSM780, Carl Zeiss).For wholemount immunofluorescence, the protocol was performed as described in (38).The 3D reconstitution of SOX9 staining have been processed with Imaris 9.1 Bitplane.

Image post-treatments
Image post-treatments were performed on Z-stack maximum intensity projection using a homemade semi-automated macro on ImageJ software (National Institutes of Health) available on Zenodo (33,34).

Quantification of RUNX1 and NR2F2 positive cells.
Gonadal Region Of Interest (ROI) was drawn manually, and nuclei were segmented upon DAPI staining using Versatile (fluorescent nuclei) model from Stardist Deep Learning plugin (39).Thresholds defining RUNX1 of NR2F2 cell positiveness were adjusted and applied to each nuclei area defined by Stardist to obtain the number of cells positive for RUNX1, NR2F2, double positive, or double negative (33) (Fig. S4A).For each genotype, 2 gonads of 3 or 4 biological replicates were analyzed.Statistical significance was determined with Student's unpaired two-tailed t-test (GraphPad Prism v.7.0b).* indicates p-value < 0.05; ns indicates p-value > 0.05.

Quantification of WT1 signal intensity
Gonadal WT1 signal was determined as the WT1 signal colocalized with GATA4 immunostaining (GATA4-positive cells allow manual surrounding of the gonad) and stored in region of interest (ROI) manager as gonad ROI (Fig. S6D).Automatic mean threshold adjustment was performed on WT1 signal intensity and small object rejected using analyze particles filter (10-infinity).The mean signal intensity within the gonad ROI was quantified after threshold adjustment.The average takes into account the number of selected pixels and therefore the area.The comparison between each set of experiments was performed by adjusting the WT1 signal intensity of the XY control gonad in each set to the same level (34).For each genotype, 2 sections and 4 biological replicates.Statistical significance was determined with Student's unpaired two-tailed t-test (GraphPad Prism v.7.0b).ns indicates p-value > 0.05.

Image processing
The DAPI/Hoechst staining marked the nuclei and was adjusted to visualize the tissues and may vary between samples.However, for the immunostaining analysis, the exposure time of the acquisition of the fluorescent signal was identical in the same experiment to allow comparison between wild type and mutants.Images were assembled using the open-source software platform OMERO (https://www.openmicroscopy.org/omero/).

Quantitative PCR analysis
Individual gonads were dissected from the mesonephros in PBS and immediately frozen in liquid nitrogen and kept at -80 °C.RNA was extracted by the RNeasy Micro Kit (Qiagen) and reverse transcribed by the MMLV reverse transcriptase (Promega).The cDNA was used as a template for quantitative PCR analysis using the SYBR Green I Master (Roche) and a LightCycler 480 System (Roche).Primer sequences are listed in Table S2.All biological replicates of different genotypes (n=3-5) are representative of independent duplicate technical replicates.Statistical significance was determined with Student's unpaired two-tailed t-test (GraphPad Prism v.7.0b).* indicates pvalue < 0.05; ns indicates p-value > 0.05.
Single-cell RNA transcriptomic analysis of XY gonads collected at E11.5 XY wild-type gonads were dissected from C57BL/6J embryos at E11.5 (21ts).Tissue was digested in 450 µL of 0.05% Trypsin and 50 µL of 2.5% collagenase and incubated for 8 minutes at 37℃.Digestion reaction was quenched with 200 µL of PBS-FBS 3%, and genital ridges were disaggregated by gently pipetting.Cells were passed through a 30 µM cell strainer into a tube to obtain single cell suspension.MoFlo XDP fluorescence-activated cell sorting (FACS) was used to isolate single cells into a chilled 96-well plate pre-loaded with 12.5 µL of CDS sorting solution (SMARTseq HT Takara Bio cat.# 634437).Following cDNA synthesis and amplification with SMARTseq HT kit, cDNA quality was assessed by Tapestation.A total of 49 cells out of 60 passed the quality checks, and indexed libraries were prepared using Nextera XT DNA Sample Preparation kit and multiplexed.RNA-Seq libraries were sequenced as 100 bp paired-end reads using the Illumina HiSEQ4000 platform at a depth of 10M reads per cell.Reads were aligned to the Ensembl mouse reference GRm38 (release 89) using STAR (v2.5.2a) with default parameters (40).Gene level quantification was performed using RSEM (v1.3.0)(41), and junction counts corresponding to the region of interest were extracted using BEDTools (v 2.27.1) (42).To quantify the KTS splice variants, we used uniquely mapped reads and calculated the percent spliced in (PSI) as the fraction of reads mapping to the +KTS or -KTS variants divided by the sum of both.
Single-cell RNA transcriptomic analysis of wildtype, -KTS KO, and +KTS KO gonads E12.0 (23±3ts) gonads from wild-type, -KTS KO, and +KTS KO mice (Table S1) were enzymatically dissociated for 10 min at 37°C using trypsin-EDTA 0.05% (Gibco, Fisher Scientific, Hampton, NH).After centrifugation, single cells were loaded on a 10× Chromium instrument (10× Genomics, Pleasanton, CA).Single-cell RNA sequencing libraries were prepared using the Chromium Single Cell 3′, version 3, reagent kit (10× Genomics), according to the manufacturer's protocol.Each genotype was performed in two independent replicates.The library quantification and quality assessment were performed using an Agilent Bioanalyzer 2100 with a high sensitivity DNA chip (Agilent Technologies, Santa Clara, CA).The libraries were diluted, pooled, and sequenced using an Illumina HiSeq4000 using paired-end 29 × 101 bp as the sequencing mode.
Fastq files were processed with CellRanger (v6.0), and the resulting count matrices were aggregated with the Read10X function implemented in Seurat (v4.0.1) (43).Doublets were filtered out independently in each individual matrix by using the DoubletFinder R package (v.2.0.2) (44) We next filtered out low-quality cells by retaining cells that expressed ≥1000 genes and had mitochondrial content ≤10%.Data were normalized using the NormalizeData and the SCT function implemented into Seurat.The top-3000 most varying genes were used to perform a principal component analysis with the RunPCA function implemented in Seurat.Cells were then clustered by using Seurat graph-based clustering (FindNeighbors and FindClusters functions) on the top-50 principal components, with default parameters.Finally, we used the Uniform Manifold Approximation and Projection (UMAP) method (RunUMAP function) to project cells in a 2D space.Cell clusters were annotated using a set of known marker genes.The FindMarkers function implemented in Seurat was used to identify significantly differentially expressed genes (DEGs) (FDR adjusted p-value ≤ 0.05) between wildtype pre-granulosa cells (belonging to cell clusters c5 and c25) to XX and XY +KTS KO pre-granulosa cells (c10, c33), and between wildtype Sertoli cells (c12) to XX and XY +KTS KO pre-granulosa cells (c10, c33).The resulting set of DEGs was further partitioned into 9 clusters by using the kmeans algorithm graphically represented as a heatmap with color-coding as shown in Fig. S7 using the pheatmap package implemented in R. Gene ontology (GO) and pathway enrichment analysis was conducted for each gene expression cluster using the AMEN suite of tools ( 45) with an BH-adjusted p value of ≤ 0.05.The FindAllMarkers function implemented in Seurat was used to identify significantly differentially expressed genes (DEGs) (FDR adjusted p-value ≤ 0.05) between cell clusters in Fig. S8.

Quantification of alternative splice variants +KTS and -KTS in single-cell transcriptomic dataset of supporting cell lineage
To calculate the usage of the +KTS and -KTS transcript along gonadal development in both sexes, we used the single-cell RNA-sequencing data described in (8, 46) (GEO GSE97519 and GSE119766, respectively).Briefly, the data were obtained from Nr5a1 expressing cells of embryonic gonads of both sexes using the C1 autoprep system from Fluidigm at different time points of the gonadal development (E10.5, E11.5, E12.5, and E13.5).Their transcriptomes were mapped on the mouse reference genome (GRCm38.p3)and the genome annotation from GENCODE (version M4, modified to integrate the GFP transgene).Cells were then clustered according to their transcriptomic profiles using the HCPC clustering method from FactoMineR R package, and the cellular identity of each cluster was assessed by the co-expression of known gonadal cell marker genes.Cell lineage reconstruction was performed using Slingshot and diffusion map from the Destiny R package to model the trajectory of the cell differentiation.To quantify the -KTS transcripts in each cell, we postulated that the exon 9 of Wt1 is a constitutive exon, that is always present in the Wt1 transcript.The quantity of +KTS transcripts was calculated by comparing the proportion of reads covering the KTS sequence compared to the rest of exon 9. To proceed, we extracted the reads of the Wt1 gene locus from the BAM files using Samtools and only kept the uniquely mapped and properly paired reads.For each cell we calculated the mean depth of coverage (the number of times a nucleotide was read during sequencing) of the nucleotides of KTS and exon 9 of Wt1 using the coverage option from Samtools.We selected the cells having at least 5 reads covering the KTS sequence and/or exon 9 to discard cells in which the exon quantification was too low and considered low confidence.Finally, we obtained the amount of +KTS and -KTS transcripts per cell by calculating the ratio of coverage of the KTS sequence compared to the exon 9.A cell with no coverage (no reads) on the KTS sequence was considered as devoid of +KTS (0%).Conversely, a cell having as much or more coverage on the KTS sequence than on exon 9 is considered having 100% of the +KTS.To estimate the expression of the respective +KTS and -KTS variants in each cell, we reported the percentage of +KTS and -KTS to the RPKM values previously calculated (8).
To follow the proportion of +KTS variants in the supporting cell lineage as they differentiated into either Sertoli or pre-granulosa cells, we selected the cells of the supporting cell lineage from their multipotent progenitor state at E10.5 until their differentiated state at E13.5, as previously defined (8).We calculated the mean percentage as well as the mean expression of +KTS and -KTS variants of the supporting cells at each stage.Plots were generated using R version 4.2 and the Ggplot2 library.Heatmap showing the expression of the top 1,189 differentially expressed genes from Sertoli cells of XY wildtype (cluster 12, see Fig. 1D-E), XY and XX +KTS KO pre-granulosa cells (c10, c33) and wildtype pre-granulosa cells (c5, c25).These genes were classified by expression patterns (k1-k9) (Data S2).Known marker genes and significantly enriched biological processes (Data S3) are shown for each expression pattern on the right-hand side.The dendrogram between the indicated genotypes shows the similarity of +KTS KO pre-granulosa cells and wildtype pre-granulosa cells.
The grayscale intensity (from white to black) indicates the averaged normalized expression of a given gene within a cluster.

Fig. S8. Differential gene expression analysis between XX -KTS KO pre-supporting cells or 860
XX +KTS KO pre-granulosa cells and XX pre-granulosa cells at E12.0.(A) Volcano plots of differential gene expression in XX -KTS KO pre-supporting cells (including 1616 XX -KTS KO cells from cluster c3, 4 from c15 and 1 from c31) or (B) XX +KTS KO pregranulosa cells (including 233 XX +KTS KO cells from c33, 59 from c10, 5 from c5 and 2 from c25) and XX pre-granulosa cells in XX control gonads (including 3535 XX control cells from c5, 865 860 from c25, 10 from c10 and 1 from c33).Red dots correspond to genes deregulated more than 1.2-fold.In XX -KTS KO pre-supporting cells, the expression of 319 genes was significantly deregulated (FDR adjusted p-value ≤ 0.05) (Data S4).S1.Details of the samples collected for the single-cell RNA sequencing.
The first column lists the identification of the samples.For each embryo, both gonads dissected from the mesonephros were collected.In -KTS KO samples, gonads of two to three embryos were pooled together.The second column lists the genotype of the samples, the third column lists the number of tail somites (ts), and the last column lists the number of cells after removal of low 890 quality ones and doublets.
Figure S2XY XY -KTS KO XX Figure S3 Fig. S4.-KTS is necessary for sex differentiation of the supporting cells.(A) Quantification of RUNX1+ and NR2F2+ cells normalized to DAPI+ cells in XY and XX wildtype and XY and XX -KTS KO gonads.n=3-4 biological replicates, 2 gonads/embryo.Data are shown as means ± SEM. (B) Immunodetection of the pre-supporting and pre-granulosa cell marker RUNX1 (magenta) and the progenitor marker NR2F2 (green) positive cells and image processing steps used for RUNX1 and NR2F2 positive cells quantification shown in a XX gonad as described in materials and methods.Scale bars: 100 μm.Nuclei labelled with DAPI are shown in gray.Upper panel: original images; lower panel: corresponding processed images: manual drawing of gonad outline on DAPI channel to determine the Region Of Interest of the gonad (left panel), threshold adjustment of RUNX1 positive (magenta) and negative (gray) cells (second panel), threshold adjustment of NR2F2 positive cells (green) and negative (gray) cells (third panel), and combination of RUNX1 (magenta), NR2F2 (green), double positive (yellow) cells, and double negative (gray) cells (last panel).